JPWO2007099711A1 - Differentiation inducer - Google Patents

Abstract

To provide an agent for inducing differentiation of bone marrow cells into endothelial cells. An agent for inducing differentiation of bone marrow cells into endothelial cells, comprising heparin or a salt thereof as an active ingredient.

Description

  The present invention relates to a differentiation inducer, particularly a differentiation inducer from bone marrow cells to endothelial cells.

Endothelial cells are the cells that form the inner walls of blood vessels and lymphatic vessels. Among these, vascular endothelial cells are located at the contact points between blood and blood vessel walls and play a role in controlling the permeability of substances, as well as between blood and tissues. Is responsible for the communication of information. These endothelial cells are known to be damaged by diseases such as hyperlipidemia, hypertension, and diabetes, and research on repair methods has been conducted.
In particular, hepatic sinusoidal endothelial cells make an important contribution to the expression of liver function through immune responses, inflammation control, microcirculation control, substance exchange, and the like. Sinusoidal endothelial cells are extremely vulnerable to hypoxia and damaging factors, and their dysfunction and loss due to cell death are associated with major liver diseases such as chronic hepatitis, cirrhosis, fulminant liver failure, liver cancer, and graft failure after transplantation. It plays a central role in the pathogenesis mechanism (Non-Patent Documents 1 to 3).
On the other hand, bone marrow cells have been suggested to be the origin of immature cells in various organs (Non-Patent Documents 4 to 6). Recently, in tissues such as liver, they are differentiated from transplanted bone marrow cells and regenerated. (Non-Patent Documents 7 to 9).
From these backgrounds, if it is possible to regenerate damaged sinusoidal endothelial cells orthotopically by using differentiation induction of bone marrow-derived cells, cirrhosis, fulminant hepatitis, etc. for which there is currently no effective treatment Expected to be a breakthrough therapy for intractable liver disease.

  On the other hand, heparin is a heteropolysaccharide having a complex structure that exists in the liver, small intestine, lung, skin, etc., and has strong blood anticoagulant activity, so that treatment of generalized intravascular blood coagulation syndrome (DIC), Treatment and prevention of various thromboembolism (venous thrombosis, myocardial infarction, pulmonary embolism, cerebral embolism, limb arterial thromboembolism, intraoperative and postoperative thromboembolism, etc.), hemodialysis, cardiopulmonary bypass It is used to prevent blood coagulation when using an extracorporeal circulation device such as when inserting a blood vessel catheter or at the time of blood transfusion and blood test. Further, it has been reported that administration of heparin increases the expression of hepatocyte growth factor (HGF), which is a growth factor of hepatocytes and sinusoidal endothelial cells, and promotes liver regeneration (Non-Patent Documents 10 to 12). ). However, in normal liver regeneration, only replication of existing hepatocytes is performed, differentiation from bone marrow cells is rare, and there is no known relationship with bone marrow cell differentiation.

Takei Y, et al. Targeted gene delivery to sinusoidal endothelial cells: DNA nanoassociate bearing hyaluronan-glycocalix. FASEB J, 2004; 18: 699-701. (10.1096 / fj.03-0494fje). Bach FH, Robson SC, Winkler H, et al. Barriers to xenotransplantation. Nature Med 1995; 1: 869-873. Mochida, S.M. Ogata, I .; , Hirata, K .; Ohta, Y .; Yamada, S .; , And Fujiwara, K .; (1990) Production of massive hepatic necrosis by endotoxin after partial hepatometry in rats. Gastroenterology 99, 771-777 Krause DS, et al. Cell 2001; 105: 369-77. Tanaka R, et al. Neuroscience 2003; 117: 531-9. Jackson KA, et al. J Clin Invest 2001; 107: 1395-402. Fujii H, et al. J Hepatol 2002; 36: 653-9. Ferry N, et al. J Hepatol 2002; 36: 659-7. Grompe M.M. Semin Liver 2003; 23: 363-72. Matsumoto K, et al. Biochem Biophys Res Commun 1996; 227: 455-61. Nakao T, et al. J Hepatol 2002; 37: 87-92. Ishii T, et al. J Biochem (Tokyo) 1995; 117: 1105-12.

  Accordingly, an object of the present invention is to provide an agent for inducing differentiation of bone marrow cells into endothelial cells.

  As a result of intensive studies, the present inventors have found that heparin or a salt thereof conventionally used as a blood coagulation inhibitor or the like remarkably promotes differentiation induction from bone marrow cells to endothelial cells.

  That is, the present invention provides an agent for inducing differentiation of bone marrow cells into endothelial cells containing heparin or a salt thereof as an active ingredient; and an effective amount of heparin or a salt thereof is administered, which is characterized by administration of an effective amount of heparin or a salt thereof. A method for inducing differentiation into cells is provided; the use of heparin or a salt thereof for the production of an agent for inducing differentiation from bone marrow cells into endothelial cells is provided.

  Since heparin or a salt thereof has an effect of inducing differentiation from bone marrow cells to endothelial cells, it can be used as an active ingredient of an agent for inducing differentiation from bone marrow cells to endothelial cells. By using the differentiation-inducing agent of the present invention, in vitro differentiation from bone marrow cells to endothelial cells can be performed efficiently, and by in vivo administration, regeneration of organs with impaired endothelial cells can be achieved. Can be provided. The differentiation-inducing agent of the present invention can provide an excellent differentiation-inducing effect on endothelial cells, particularly by transplanting bone marrow cells into a target tissue developing a disease such as inflammation.

It is the HE (hematoxylin eosin) stained image (FIG. 1a) and the Azan stained image (FIG. 1b) of the liver lobule of the carbon tetrachloride administration group. It is an anti-GFP antibody dyeing | staining image of the liver lobule of a carbon tetrachloride administration group. FIG. 3 shows anti-GFP antibody-stained images (FIG. 3a, d), anti-CD31 antibody-stained image (FIG. 3c), and superimposed image (FIG. 3b) of liver lobule in the control group. FIG. 4 shows an anti-GFP antibody-stained image (FIG. 4a, d), an anti-CD31 antibody-stained image (FIG. 4c), and a superposed image (FIG. 4b) of liver lobules in the carbon tetrachloride administration group. FIG. 5 shows an anti-GFP antibody-stained image (FIG. 5a, d), an anti-CD31 antibody-stained image (FIG. 5c), and a superimposed image (FIG. 5b) of the liver lobule in the carbon tetrachloride + dalteparin sodium administration group. It is the graph which showed the comparison in each group of GFP + CD31 + cell number.

  The heparin used in the differentiation-inducing agent of the present invention is obtained, for example, from the liver, lung or intestinal mucosa of a healthy edible animal (eg, cow, pig, etc.), and uronic acid and glucosamine are alternately linked to 1,4. Examples thereof include sulfated mucopolysaccharides having a structure and an average molecular weight of 5,000 to 20,000. Further, as heparin, low molecular weight heparin is preferable from the viewpoint that excellent differentiation induction efficiency is obtained. Examples of the low molecular weight heparin include those having an average molecular weight of 2000 to 10,000. Low molecular weight heparin has high safety because of low risk of bleeding and the like, and has an excellent differentiation induction promoting activity, so the average relative molecular weight is about 5000 and 90% is distributed in the molecular weight range of 2000 to 9000. Dalteparin having a degree of sulfate esterification of 2 to 2.5 per disaccharide is preferred. Examples of heparin salts include sodium salts and calcium salts.

  Heparin has already been developed as a raw material for pharmaceuticals and cosmetics, and a commercially available product can be used. However, heparin can be prepared by a method according to the method disclosed in the 14th Japanese Pharmacopoeia. Low molecular weight heparin can be obtained by decomposing heparin derived from bovine or porcine intestinal mucosa with hydrogen peroxide and cupric sulfate.

  As shown in Examples below, heparin induced differentiation from bone marrow cells to endothelial cells in carbon tetrachloride liver injury model mice. Therefore, heparin can be used as an active ingredient of an agent for inducing differentiation from bone marrow cells to endothelial cells. Conventionally, heparin has been reported to increase the expression of hepatocyte growth factor (HGF), thereby proliferating non-parenchymal cells such as liver parenchymal cells and sinusoidal endothelial cells. However, in normal liver regeneration, only replication of existing hepatocytes is performed, differentiation from bone marrow cells is rare, and there is no known relationship with the differentiation of bone marrow cells. Inducing differentiation is surprising.

The differentiation-inducing agent of the present invention is used for inducing differentiation from bone marrow cells in vivo as well as bone marrow cells separated from the living body and cultured in an in vitro system, but suitable for inducing differentiation of bone marrow cells in vivo. Used for.
In addition, the differentiation inducer of the present invention can be used for induction of differentiation from bone marrow cells inherent in a living body and differentiation induction of bone marrow cells transplanted into a target tissue, and among these, high differentiation induction efficiency can be obtained. In view of the excellent therapeutic effect, it is preferably used for inducing differentiation of bone marrow cells transplanted into the target tissue. In this case, the differentiation inducer of the present invention is preferably administered with heparin or a salt thereof after transplantation.
The differentiation-inducing agent of the present invention is suitably used when the target tissue to which bone marrow cells are transplanted is the liver, because superior differentiation-inducing efficiency can be obtained with heparin or a salt thereof. An organ into which bone marrow cells are transplanted is preferably used when a disease such as inflammation has developed. When the organ is the liver, examples of the disease include hepatitis such as fulminant hepatitis and chronic hepatitis, end-stage liver disease such as cirrhosis, and liver cancer, which are preferably used when hepatitis develops. When used for a liver that has developed hepatitis, the differentiation-inducing agent of the present invention can also induce differentiation of the liver into sinusoidal endothelial cells. Although bone marrow cells transplanted into the liver have been reported to differentiate into hepatocytes, etc., differentiation into sinusoidal endothelial cells can also be induced by the differentiation inducer of the present invention.

  Examples of the subject to which the differentiation inducer of the present invention is used include mammals such as humans, mice, and rats.

  The differentiation inducer of the present invention is used in the form of a general pharmaceutical preparation, and typical examples thereof include injections (solutions, suspensions, etc.) and the like.

  When prepared as injections, solutions, emulsions and suspensions are preferably sterilized and isotonic with blood, and when used in these forms, those commonly used in this field as diluents, For example, water, ethanol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used. In this case, the amount of sodium chloride, glucose or glycerin necessary to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a normal solubilizing agent, buffering agent, soothing agent, etc. may be added. Also good.

  The dosage of these pharmaceutical preparations of the present invention is appropriately selected depending on the usage, patient age, gender, disease severity and other conditions. Usually, the mass of heparin is about 5 I.D per kg body weight per day. U. / Kg-100I. U. / Kg is preferably injected for 1 to 24 hours.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Reference Example 1 Preparation of GFP-positive bone marrow cells 8-week-old GFP transgenic mice (Okabe M, et al. FEBS Lett 1997; 407: 313-9) were treated with 5-fluorouracil (150 μg / g BW) and proliferating cells. Destroyed. After 48 hours, the limbs were sacrificed by cervical dislocation and the extremities were removed, and then washed with Dulbecco's modified Eagle medium (GIBCO, Grand Island, New York) using a 28G needle to obtain GFP-positive bone marrow cells from the femur and cervical bone marrow cavity. .

Example 1 Preparation of hepatic lobule tissue from bone marrow transplanted hepatitis-caused mice [Bone marrow transplantation to hepatitis-caused mice]
Female 8-week-old C57 / B6 mice (Nippon Charles River Co., Ltd.) were administered 0.2 ml of carbon tetrachloride per kg of the individual at a frequency of twice a week for 4 weeks. After irradiating a whole body of mice after carbon tetrachloride administration for 4 weeks with a lethal dose of X-rays (10 Gy), 1 × 10 ⁷ GFP-positive bone marrow cells obtained in Reference Example 1 were tailed with a 31 G needle. Injection into a vein and bone marrow transplantation were performed. Dalteparin sodium 50 IU / kg was administered intraperitoneally for 28 days to the group of mice subjected to bone marrow transplantation from the next day of bone marrow transplantation to obtain a carbon tetrachloride + dalteparin sodium administration group. Mice that did not receive dalteparin sodium were treated with carbon tetrachloride. In addition, bone marrow transplantation was performed by injecting 1 × 10 ⁷ GFP-positive bone marrow cells into C57 / B6 mice not charged with carbon tetrachloride in the same manner as above, and used as a control group.
(Preparation of liver lobule tissue)
For each group of mice, the liver was perfused via the heart with a PBS (phosphate buffered saline) buffer, and blood cells contaminated in the liver were removed. Perfused liver was fixed overnight with 4% paraformaldehyde and then treated with 30% sucrose for several days. The obtained liver tissue was frozen with powder dry ice, and was cut into 18 μm tissue sections using a cryostat (LEICA CM 1900, FINETEC).
Representative micrographs of HE (hematoxylin and eosin) -stained images and Azan-stained images of tissues of the carbon tetrachloride administration group are shown in FIGS. 1a and 1b, respectively. As shown in FIG. 1a, no infiltration of inflammatory cells was observed, but as shown in FIG. 1b, fibrosis of liver tissue was observed, confirming that liver damage had occurred.

Example 2 Observation of GFP-positive cells and CD31-positive cells An anti-GFP antibody-stained image and an anti-CD31 antibody-stained image of the obtained tissue were observed. That is, the obtained tissue section was washed 3 times with PBS buffer and then immersed in 10% goat serum for 1 hour. After the serum was washed, it was treated with an anti-GFP antibody (Santa Cruz Biotechnology) for 1 hour, and then a TRITC goat anti-IgG antibody (Santa Cruz Biotechnology) was cultured at 37 ° C. for 1 hour and treated with a secondary antibody. Microscopic observation was performed on a slide glass on which 1% gelatin / saline solution was placed. An anti-CD31 antibody stained image was also obtained in the same manner except that an anti-CD31 antibody (BD Bioscience) was used instead of the anti-GFP antibody.

[Control group]
A stained image of the tissue from the control group is shown in FIG. 3a and 3d show anti-GFP antibody stained images. In addition, an anti-CD31 antibody (endothelial cell marker) stained image in the region shown in FIG. 3d is shown in FIG. 3c. FIG. 3b is an image obtained by superimposing these anti-GFP antibody stained image and anti-CD31 antibody stained image. As shown in these figures, GFP-positive cells were slightly observed, but GFP-positive and CD31-positive cells were hardly seen.

[Carbon tetrachloride administration group]
The anti-GFP antibody dyeing | staining image of the structure | tissue in a carbon tetrachloride administration group is shown to FIG. 2, FIG. 4a, FIG. As shown in FIG. 2, FIG. 4a, and FIG. 4d, most of the GFP positive cells in the hepatic lobule spread in a network structure from the portal vein region to the central vein region. Not observed. This suggests that GFP positive cells are closely related to sinusoidal endothelial cells.
Also, an anti-CD31 antibody staining image as an endothelial cell marker is shown in FIG. 4c, and an image superimposed with an anti-GFP antibody staining image (FIG. 4d) in the same region is shown in FIG. 4b. The arrows in FIG. 4b indicate cells that are GFP positive and CD31 positive. As shown, most of the GFP positive cells were CD31 positive. From this, it is considered that the transplanted bone marrow cells specifically differentiated into sinusoidal endothelial cells.

[Carbon tetrachloride + dalteparin sodium administration group]
FIG. 5 shows an anti-GFP antibody stained image and an anti-CD31 antibody stained image of the tissue in the carbon tetrachloride administration group. 5a and 5d are anti-GFP antibody stained images, FIG. 5c is an anti-CD31 antibody stained image, and FIG. 5b is an image obtained by superimposing FIGS. 5d and 5c. As shown in these, a significant increase in GFP positive cells was observed compared to the carbon tetrachloride administration group. Most of the GFP positive cells were CD31 positive.

[Quantitative comparison of differentiation induction efficiency]
Based on the obtained stained images, the number of GFP-positive and CD31-positive cells (GFP + CD31 + cells) in the unit region was determined and compared among the above three groups (FIG. 6). As shown in FIG. 6, the control group had a very small number of 1.0 ± 1.2 per region, whereas the carbon tetrachloride administration group had a large number of 3.8 ± 1.3 per region. there were. In the carbon tetrachloride + dalteparin sodium administration group, 8.3 ± 1.3 per region, and a marked increase in the number of sinusoidal endothelial cells differentiated from bone marrow cells was observed (with the carbon tetrachloride administration group and P <0.05). From the above, it is clear that when bone marrow cells are transplanted into a liver that has developed hepatitis, it differentiates into sinusoidal endothelial cells, and the administration of dalteparin sodium significantly increases this differentiation induction efficiency. Became clear.

Claims (21)

  An agent for inducing differentiation of bone marrow cells into endothelial cells, comprising heparin or a salt thereof as an active ingredient.   The differentiation inducer according to claim 1, wherein the heparin is low molecular weight heparin.   The differentiation inducer according to claim 1 or 2, wherein the bone marrow cells are transplanted bone marrow cells.   The differentiation inducer according to claim 3, wherein heparin or a salt thereof is administered after bone marrow cells are transplanted into the target tissue.   The differentiation inducer according to claim 4, wherein the target tissue is liver.   6. The differentiation inducer according to claim 5, wherein the target tissue is a liver developing hepatitis.   The differentiation inducer according to any one of claims 1 to 6, wherein the endothelial cell is a hepatic sinusoidal endothelial cell.   A method for inducing differentiation from bone marrow cells to endothelial cells, comprising administering an effective amount of heparin or a salt thereof.   9. The differentiation induction method according to claim 8, wherein the heparin is a low molecular weight heparin.   The differentiation induction method according to claim 8 or 9, wherein the bone marrow cells are transplanted bone marrow cells.   The differentiation induction method according to claim 10, wherein heparin or a salt thereof is administered after bone marrow cells are transplanted into a target tissue.   The differentiation induction method according to claim 11, wherein the target tissue is a liver.   The differentiation induction method according to claim 12, wherein the target tissue is a liver developing hepatitis.   The differentiation induction method according to any one of claims 8 to 13, wherein the endothelial cells are hepatic sinusoidal endothelial cells.   Use of heparin or a salt thereof for the production of an agent for inducing differentiation of bone marrow cells into endothelial cells.   The use according to claim 15, wherein the heparin is a low molecular weight heparin.   The use according to claim 15 or 16, wherein the bone marrow cells are transplanted bone marrow cells.   The use according to claim 17, wherein heparin or a salt thereof is administered after bone marrow cells are transplanted into a target tissue.   The use according to claim 18, wherein the target tissue is liver.   The use according to claim 19, wherein the target tissue is a liver developing hepatitis.   The use according to any one of claims 15 to 20, wherein the endothelial cells are hepatic sinusoidal endothelial cells.